GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs) form a family of proteins that play key roles in cell adhesion and migration, tumor progression, as well as neuronal development. A body of evidence also indicates that ARFGAPs are involved in various diseases, including cancer, Alzheimer's disease, and autism. For example, ASAP1 (one ARFGAP) expression is strongly up-regulated in a variety of tumors in comparison with normal tissue and that this expression correlates with poor metastasis-free survival and prognosis in colorectal cancer patients. However, the detailed mechanism by which ARFGAPs regulate different diseases is largely unknown. Small molecule ARFGAP modulators will be invaluable tools to dissect ARFGAP-regulated cell signaling and further validate ARFGAPs as drug targets. However, there are no small molecule ARFGAP inhibitors in the literature possibly due to the fact that the currently available ARFGAP assays are not amenable to high throughput screening. In our efforts to dissect ARFGAP-regulated cell signaling, a novel fluorescence polarization-based ARFGAP assay has been developed. The Z'factor of the assay is 0.75 in 384-well format. When applied to a pilot screen of the Prestwick library of around 1,000 compounds, the assay demonstrated high reproducibility, reasonable hit rates, and suitability for automation. This proposal seeks to further develop this novel assay to screen libraries of small molecules for ARFGAP inhibitors through two specific aims: 1) Optimize assay conditions that are suitable for high throughput screen in 384-well format;and 2) Configure the assay that is developed in aim 1 for high throughput screening of small molecule library. The proposed assay for ARFGAP inhibitors will represent the first such assay that is not based on radioactivity and will likely generate ARFGAP-selective small molecule inhibitors. Given the important roles ARFGAPs played in basic cell biology, any ARFGAP inhibitor that is generated from the proposed screen will likely find broad applications in the scientific community. Furthermore, the small molecule ARFGAP inhibitors have the potential to function as lead compounds for drug development in various diseases including cancer and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under-investigated area of GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs). The proposed research has relevance to public health, because ARFGAPs are involved in various diseases, and the small molecules developed could be used as probes to understand the ARFGAP interaction network and potential lead compounds for drug discovery.